Interaction of group IA phospholipase A2 with metal ions and phospholipid vesicles probed with deuterium exchange mass spectrometry.

Deuterium exchange mass spectrometric evaluation of the cobra venom (Naja naja naja) group IA phospholipase A(2) (GIA PLA(2)) was carried out in the presence of metal ions Ca2+ and Ba2+ and phospholipid vesicles. Novel conditions for digesting highly disulfide bonded proteins and a methodology for studying protein-lipid interactions using deuterium exchange have been developed. The enzyme exhibits unexpectedly slow rates of exchange in the two large a-helices of residues 43-53 and 89-101, which suggests that these alpha-helices are highly rigidified by the four disulfide bonds in this region. The binding of Ca2+ or Ba2+ ions decreased the deuterium exchange rates for five regions of the protein (residues 24-27, 29-40, 43-53, 103-110, and 111-114). The magnitude of the changes was the same for both ions with the exception of regions of residues 24-27 and 103-110 which showed greater changes for Ca2+. The crystal structure of the N. naja naja GIA PLA(2) contains a single Ca2+ bound in the catalytic site, but the crystal structures of related PLA(2)s contain a second Ca2+ binding site. The deuterium exchange studies reported here clearly show that in solution the GIA PLA(2) does in fact bind two Ca2+ ions. With dimyristoylphosphatidylcholine (DMPC) phospholipid, vesicles with 100 mu M Ca2+ present at 0 degrees C, significant areas on the i-face of the enzyme showed decreases in the rate of exchange. These areas included regions of residues 3-8, 18-21, and 56-64 which include Tyr-3, Trp-61, Tyr-63, and Phe-64 proposed to penetrate the membrane surface. These regions also contained Phe-5 and Trp-19, proposed to bind the fatty acyl tails of substrate.